A widespread means of communication between animal cells involves the direct, passive exchange of small molecules via cell. to-cell channels. These channels, comprised of "connexins" in vertebrates, are aggregated to form gap junctions (GJs) and play a significant role in the transmission of electrical signals, the control of cell growth and the development of various organ systems. The importance of GJ communication is reflected in the number of disease states linked to junctional deficiencies and/or connexin mutations. Given the short half-lives of connexins, it is necessary to have efficient and effective mechanisms for regulating the assembly of GJs, both qualitatively and quantitatively. The goal of this laboratory is to understand the process of G3 assembly and its regulation. Our research approach is based on an integrated set of structural, functional, and molecular methods applied to cultured cells. We believe this will provide for a broad understanding of GJ assembly and its regulation. The studies proposed here will focus on enhanced GJ assembly, a process that is mediated by cAMP and that relies on the intracellular trafficking of Cx43. Although connexin trafficking represents a poorly understood aspect of cell communication, it likely to be important in both cellular physiology and pathology. We will address the following three aims: Aim # 1: Determine how the microtubule dependent delivery at Cx43-hemichannels to the plasma membrane is influenced by cAMP; cell contact and specific Cx43 mutations. Aim #2: Determine whether nonjunctional, plasma membrane hemichannels are endocytosed and recycled and whether this is regulated by a cAMP-dependent mechanism. Aim #3: Determine how membranes with Cx43-hemichannels are transported along microtubules and respond to increases in both cAMP and cell contact. Both functional (dye transfer) and structural (freeze-fracture EM) assays for GJ assembly will be used to examine fibroblasts from "knock out" mice transfected with wild type Cx43 or different mutant forms. The "restoration of dye uptake" following the inhibition of plasma membrane (PM) hemichannels with specific Cx43 antibodies, as well as the biotinylation of nonjunctional, Cx43 in the PM, will be used to measure the delivery of hemichannels to the PM. To analyze transport in living ceils, we will use Cx43-GFP (green fluorescence protein) chimeras. We will also employ newly developed methods tar tagging Cx43 with tetracysteine for fluorescence studies in living cells and for studies at the EM level. Finally, "pull-down" experiments will identify proteins associating with Cx43, in response to cAMP, and various biochemical techniques will be used to study wild type and mutant Cx43 in fibroblasts.